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Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections 1 . Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes 2 . The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures. Detailed atomic models of axonemes from algal flagella and human respiratory cilia, which are hair-like protrusions from cells that enable motility and clear mucus from human airways, could provide insights into how they function.

Background Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder resulting from pathogenic variants in three distinct genes, with most of the variants occurring in the electron transfer flavoprotein-ubiquinone oxidoreductase gene ( ETFDH) . Recent evidence of potential founder variants for MADD in the South African (SA) population, initiated this extensive investigation. As part of the International Centre for Genomic Medicine in Neuromuscular Diseases study, we recruited a cohort of patients diagnosed with MADD from academic medical centres across SA over a three-year period. The aim was to extensively profile the clinical, biochemical, and genomic characteristics of MADD in this understudied population. Methods Clinical evaluations and whole exome sequencing were conducted on each patient. Metabolic profiling was performed before and after treatment, where possible. The recessive inheritance and phase of the variants were established via segregation analyses using Sanger sequencing. Lastly, the haplotype and allele frequencies were determined for the two main variants in the four largest SA populations. Results Twelve unrelated families (ten of White SA and two of mixed ethnicity) with clinically heterogeneous presentations in 14 affected individuals were observed, and five pathogenic ETFDH variants were identified. Based on disease severity and treatment response, three distinct groups emerged. The most severe and fatal presentations were associated with the homozygous c.[1067G > A];c.[1067G > A] and compound heterozygous c.[976G > C];c.[1067G > A] genotypes, causing MADD types I and I/II, respectively. These, along with three less severe compound heterozygous genotypes (c.[1067G > A];c.[1448C > T], c.[740G > T];c.[1448C > T], and c.[287dupA*];c.[1448C > T]), resulting in MADD types II/III, presented before the age of five years, depending on the time and maintenance of intervention. By contrast, the homozygous c.[1448C > T];c.[1448C > T] genotype, which causes MADD type III, presented later in life. Except for the type I, I/II and II cases, urinary metabolic markers for MADD improved/normalised following treatment with riboflavin and L-carnitine. Furthermore, genetic analyses of the most frequent variants (c.[1067G > A] and c.[1448C > T]) revealed a shared haplotype in the region of ETFDH , with SA population-specific allele frequencies of < 0.00067–0.00084%. Conclusions This study reveals the first extensive genotype–phenotype profile of a MADD patient cohort from the diverse and understudied SA population. The pathogenic variants and associated variable phenotypes were characterised, which will enable early screening, genetic counselling, and patient-specific treatment of MADD in this population.

Primary ciliary dyskinesia (PCD) is associated with abnormal organ positioning (situs) and congenital heart disease (CHD). This study investigated genotype–phenotype associations in PCD to facilitate risk predictions for cardiac and laterality defects. This retrospective cohort study of 389 UK patients with PCD found 51% had abnormal situs and 25% had CHD and/or laterality defects other than situs inversus totalis. Patients with biallelic mutations in a subset of nine PCD genes had normal situs. Patients with consanguineous parents had higher odds of situs abnormalities than patients with non-consanguineous parents. Patients with abnormal situs had higher odds of CHD and/or laterality defects.

RationalePrimary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal.ObjectivesTo determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive.MethodsNext-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay.ResultsSixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed.ConclusionsThe CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.

To describe our experience with a large cohort (411 patients from 288 families) of various forms of skeletal dysplasia who were molecularly characterized. Detailed phenotyping and next-generation sequencing (panel and exome). Our analysis revealed 224 pathogenic/likely pathogenic variants (54 (24%) of which are novel) in 123 genes with established or tentative links to skeletal dysplasia. In addition, we propose 5 genes as candidate disease genes with suggestive biological links (WNT3A, SUCO, RIN1, DIP2C, and PAN2). Phenotypically, we note that our cohort spans 36 established phenotypic categories by the International Skeletal Dysplasia Nosology, as well as 18 novel skeletal dysplasia phenotypes that could not be classified under these categories, e.g., the novel C3orf17-related skeletal dysplasia. We also describe novel phenotypic aspects of well-known disease genes, e.g., PGAP3-related Toriello–Carey syndrome–like phenotype. We note a strong founder effect for many genes in our cohort, which allowed us to calculate a minimum disease burden for the autosomal recessive forms of skeletal dysplasia in our population (7.16E-04), which is much higher than the global average. By expanding the phenotypic, allelic, and locus heterogeneity of skeletal dysplasia in humans, we hope our study will improve the diagnostic rate of patients with these conditions.

Primary congenital glaucoma (PCG) accounts for 26–29% of childhood blindness in Egypt. The identification of disease causing mutations has not been extensively investigated. We aimed to examine the frequency of CYP1B1 and MYOC mutations in PCG Egyptian patients, and study a possible genotype/phenotype correlation. Ninety-eight patients with PCG diagnosed at the Ophthalmology department of Alexandria Main University Hospital were enrolled. Demographic and phenotypic characteristics were recorded. Patients and 100 healthy subjects (control group) were screened for two mutations in CYP1B1 gene (G61E, R368H) and one mutation in MYOC gene (Gln48His) using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP). Phenotypic characteristics pertaining to disease severity were compared. Nineteen patients (19%) with PCG were found positive for one or more of the mutations screened for. Seven patients (7%) were homozygous for the G61E mutation. Ten patients (10%) were heterozygous; 6 for the G61E mutation, 2 for the R368H mutation and 2 for the Gln48His mutation. Two patients (2%) were double heterozygotes harboring a R368H as well as a Gln48His mutation. The most common mutation observed was the G61E in 13 patients; 7 homozygotes and 6 heterozygotes for the mutation. The control group were negative for all mutations screened for. No significant correlations between the mutations and phenotype severity were detected. A statistically significant positive correlation however was found between the different mutations and each of the IOP and the cup/disk ratio. The current study further endorses the role of CYP1B1 mutations in the etiology of PCG among Egyptian patients and is the first study to report MYOC gene mutation in Egyptian patients with PCG.

We combined deep phenotype data and whole-genome sequencing results from the UK 100,000 Genomes Project (100KGP) to characterize the genetic spectrum of infantile nystagmus and albinism, and to explore genotype–phenotype correlations in this cohort. Participants were enrolled in the study based on the clinical codes for albinism or infantile nystagmus. Whole genome sequencing data was retrieved and variants in known nystagmus/albinism genes (PanelApp R39 panel) were analysed alongside genome-wide findings. We ascertained 473 affected individuals (388 families). Positive genetic findings were obtained in 218 participants (46%), including 176 (37%) with definitive diagnoses. Pathogenic variants were found in 16 of 38 panel genes, most commonly in TYR (56 families) and OCA2 (21 families). Recurrent variants (24% of 103 different variants) were identified in six genes. An additional 36 off-panel genes accounted for diagnoses in 45 families, including four dual genetic diagnoses in three families. Phenotypically, refractive errors and foveal hypoplasia-related diagnoses were significantly enriched (odds ratio ~2.8 for refractive disorders). Strabismus and neurodevelopmental features were also over-represented in the cohort. We show that whole-genome sequencing provided a high diagnostic yield in infantile nystagmus/albinism and uncovered a broad allelic spectrum. Integrating comprehensive phenotype data identified distinct genotype–phenotype relationships.

BackgroundPrimary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests.MethodsThe diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries.ResultsMost (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results.ConclusionsThis study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi‐allelic pathogenic variants in BLM , encoding a nuclear‐binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult‐onset mitochondrial disease resulting from bi‐allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory‐motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom‐like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult‐onset mitochondrial disease and more severe variants causing a Bloom‐like syndrome with mitochondrial dysfunction in childhood. Synopsis We report cases of adult‐onset mitochondrial disease causes by pathological variants in the topoisomerase TOP3A, which localises both to mitochondria and the nucleus. We propose that different variants result in different disease outcomes related to the mitochondrial or nuclear forms of the enzyme. Pathological variants in TOP3A cause adult‐onset mitochondrial disease. Pathological TOP3A variants cause mitochondrial genome instability and muscle mitochondrial dysfunction. TOP3A variants affect the DNA binding, relaxation and ssDNA decatenation functions of the protein, and impair mtDNA replication. The severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants associated with mitochondrial disease and more severe variants associated with Bloom syndrome. Graphical Abstract We report cases of adult‐onset mitochondrial disease caused by pathological variants in the topoisomerase TOP3A, which localises both to mitochondria and nucleus. We propose that different variants result in different disease outcomes related to the mitochondrial or nuclear forms of the enzyme.

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into fully differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalised investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in the MCIDAS gene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patient's nasal epithelial cells was seen in vitro, suggesting a novel avenue for drug evaluation and development in PCD.